Development of an Agrobacterium-mediated CRISPR/Cas9 gene editing system in jute (Corchorus capsularis)

Abstract

Jute (Corchorus capsularis L.) is the second most important natural plant fiber source after cotton. However, developing an efficient gene editing system for jute remains a challenge. In this study, the transgenic hairy root system mediated by Agrobacterium rhizogenes strain K599 was developed for Meifeng 4, an elite jute variety widely cultivated in China. The transgenic hairy root system for jute was verified by subcellular localization and bimolecular fluorescence complementation (BiFC) assays. The CHLOROPLASTOS ALTERADOS 1 (CcCLA1) gene, which is involved in the development of chloroplasts, was targeted for editing at two sites in Meifeng 4. Based on this hairy root transformation, the gRNA scaffold was placed under the control of cotton ubiquitin GhU6.7 and -GhU6.9 promoters, respectively, to assess the efficiency of gene editing. Results indicated the 50.0% (GhU6.7) and 38.5% (GhU6.9) editing events in the target 2 alleles (gRNA2), but no mutation was detected in the target 1 allele (gRNA1) in transgenic-positive hairy roots. CcCLA1 gene editing at gRNA2 under the control of GhU6.7 in Meifeng 4 was also carried out by Agrobacterium tumefaciens-mediated transformation. Two CcCLA1 mutants were albinic, with a gene editing efficiency of 5.3%. These findings confirm that the CRISPR/Cas9 system, incorporating promoter GhU6.7, can be used as a gene editing tool for jute.

Headnote

ABSTRACT

Jute (Corchorus capsularis L.) is the second most important natural plant fiber source after cotton. However, developing an efficient gene editing system for jute remains a challenge. In this study, the transgenic hairy root system mediated by Agrobacterium rhizogenes strain K599 was developed for Meifeng 4, an elite jute variety widely cultivated in China. The transgenic hairy root system for jute was verified by subcellular localization and bimolecular fluorescence complementation (BiFC) assays. The CHLOROPLASTOS ALTERADOS 1 (CcCLA1) gene, which is involved in the development of chloroplasts, was targeted for editing at two sites in Meifeng 4. Based on this hairy root transformation, the gRNA scaffold was placed under the control of cotton ubiquitin GhU6.7 and -GhU6.9 promoters, respectively, to assess the efficiency of gene editing. Results indicated the 50.0% (GhU6.7) and 38.5% (GhU6.9) editing events in the target 2 alleles (gRNA2), but no mutation was detected in the target 1 allele (gRNA1) in transgenic-positive hairy roots. CcCLA1 gene editing at gRNA2 under the control of GhU6.7 in Meifeng 4 was also carried out by Agrobacterium tumefaciens-mediated transformation. Two CcCLA1 mutants were albinic, with a gene editing efficiency of 5.3%. These findings confirm that the CRISPR/Cas9 system, incorporating promoter GhU6.7, can be used as a gene editing tool for jute.

Keywords:

Jute

Agrobacterium-mediated transformation

Genome editing

Hairy root system

1. Introduction

Jute (Corchorus L.) ranks second only to cotton as an important source of plant fiber. Cultivation of jute is predominantly concentrated in Bangladesh, India, and China [1]. Jute is not only a renewable resource for biofuels but also provides valuable raw materials for the paper and textile industries [2]. The fiber has soft in texture and has a golden color, earning it the reputation of the 'golden fiber'. Jute production faces numerous biotic and abiotic challenges. Traditional breeding of a jute variety is labor-intensive and time-consuming and typically takes eight to ten generations. Considering these challenges and the urgent need for more precise breeding, we set out to develop an Agrobacterium-mediated CRISPR/Cas9 gene editing system following the release of the jute genome map in a previous study [3].

The CRISPR/Cas9 gene editing system has been used for genetic improvement of crops such as rice [3,4], wheat [5], and cotton [6,7]. However, there are few reports of its application in jute. With the gradual improvement of a genetic transformation system in jute, a CRISPR-based gene editing system will play a significant role in basic research and molecular breeding in this crop. The CHLOROPLASTOS ALTERADOS 1 (CLA1) gene encodes 1-deoxylulose 5-phosphate synthase, that is essential for synthesis of both chlorophyll and carotenoids. The CLA1 gene serves as a visual marker in development of gene editing systems due to a bleaching phenotype upon disruption by CRISPR/Cas9 [8]. The objectives of this study were to: (1) develop an efficient transgenic hairy roots induction system in jute for gene characterization and functional study, and (2) establish a CRISPR/Cas9-based gene editing system targeting the CcCLA1 gene.

2. Materials and methods

2.1. Plant materials and growth conditions

The pure line, Meifeng 4, provided by the Laboratory of Fiber Crop Genetic Breeding and Multiple Utilization at Fujian Agriculture and Forestry University, was chosen for this study. The main steps for cultivating aseptic seedlings were as follows: intact and plump seeds of Meifeng 4 were placed in a conical flask and disinfected with 75% ethanol for 30 s, followed by washing with aseptic water for 3 min. They were further disinfected with 50% 84-Disinfectant (Chlorine-containing disinfectant, Kanglipai, Fuzhou, Fujian, China) for 20 min and washed four times with aseptic water, each washing lasting 3 min. After drying on aseptic filter paper for 5 min, the seeds were placed on MS Seeds Germination Medium (SGM, Table S1) and maintained under a 16 h/8 h (light/darness) photoperiod at 26 °C until two leaves emerged, typically within 8-12 d.

2.2. Bacterial culture and media

Agrobacterium rhizogenes strain K599 was used for transient expression of hairy roots, and Agr. tumefaciens strain GV3101 was used for stable genetic transformation. Strain K599, carrying either an overexpression vector or the CRISPR/Cas9 mediated gene editing vectors (pGhU6.7/GhU6.9:gRNA-Ubi:Cas9), was used to induce hairy root formation in jute plants. A positive Agrobacterium colony was picked from Luria-Bertani (LB) solid medium supplemented with kanamycin (50 mg L*¹) and streptomycin (50 mg L*¹). One mL of LB liquid medium containing kanamycin (50 mg L-¹) and streptomycin (50 mg L*¹) was inoculated with a selected bacterial colony and cultivated at 28 °C in darkness at 200 r min¹ for 16-18 h. Subsequently, 150 mL of fresh LB liquid medium (containing kanamycin 50 mg L*¹ and streptomycin 50 mg L*¹) was inoculated with the previously cultured bacterial liquid and culturing continued for 16-20 h. The bacterial suspension, with an optical density of approximately OD600 = 0.8, was collected in a 50 mL centrifuge tube and centrifuged at 5000 r min*¹ for 10 min at 24 °C. The bacterial cells were resuspended in the infection medium (INM, Table S1) adjusted to OD600, and incubated in darkness for 2 h.

2.3. Induction of hairy roots in jute

Explants from well-grown jute seedlings (Fig. S1) were soaked in 30 mL of infection medium (INM) (OD 600 = 0.5-1.5) for 10-20 min. After soaking, the explants were placed on filter paper to remove excess moisture and transferred to a new culture dish containing co-cultivation medium (HRCM, Table S1) for 48 h at 22 °C. The explants were then rinsed three times with aseptic water containing 250 mg L*¹ Timentin (a penicillin group antibiotic) and transferred to antibacterial medium (HRAM, Table S1) for one day. Finally, the explants were rinsed three times with aseptic water containing 400 mg L*¹ Timentin, dried, and transferred to a hairy root induction medium (HRIM, Table S1) for cultivation.

Bimolecular fluorescence complementation (BiFC) was used to study protein interaction in vivo. SPL10 interacts with WOX3B in rice [9]. We generated and transformed the plasmids pCAMBIA1300S-YN-SPL10 and pCAMBIA2300S-YC-WOX3 into Agr. rhizogenes strain K599 and introduced them into jute plants with strong expression of hairy roots. Fluorescence in the roots was observed by confocal microscopy (TCS SP8, Leica).

2.4. Screening and detection of transgenic plants with hairy roots

The hairy roots were initially observed using a fluorescence microscope (Nikon Eclipse-Ni, Japan) based on green fluorescent protein (GFP) and red fluorescent protein (DsRed2) markers. Plant genomic DNA was extracted using a plant genomic DNA extraction kit (TIANGEN, LOT#Y1616). PCR amplification with specific primers (P-GFP-F/R, Table S3) for the GFP gene and sequencing of the amplified products were performed to confirm transgenic roots. PCR amplification with the specific primers (P-Cas9-F/R, Table S3) and subsequent sequencing of the amplified product was used to confirm the presence of the Cas9 gene in hairy roots or shoots regenerated from the roots. PCR amplification with primer (P-CLA1-F/R, Table S3) containing the target site was performed, potentially edited fragments were fused into the T-vector, and Sanger sequencing was used to determine whether mutation was successfully induced.

One µg of total RNA from the plants, extracted using an E.Z.Ν.Α Plant RNA Kit (Omega, LOT#R6827010000D27W169, USA), was reverse transcribed into first-strand cDNA using a reverse transcription kit (TIANGEN, LOT#Y1425). The concentration and purity of DNA or RNA were measured using a Nano Drop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA). Three independent reactions were performed for putative genes using Fast Start Universal SYBR Green Master Mix (ROX) with a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), following the manufacturer's directions. Each reaction contained cDNA (2 µL, 100 ng µL*1), gene-specific primers (1 µL, 10 µmol L*1) and 2× SYBR Green Master Mix (10 µL) in a final volume of 20 µL. The real-time quantitative PCR procedure followed Zhang et al. [3]. Three biological replicates with two technical replicates of each sample were performed to acquire reliable results. Data were recorded as means + standard deviation of three independent experiments, and analyses were conducted using SPSS Statistics 20.

2.5. Regeneration of transgenic jute plants

Previous studies showed that jute plantlets can be regenerated in vitro from explants of cotyledon node [10-12]. We optimized this regeneration system to achieve the best bud induction effect. Explants were cut from jute seedlings (Fig. S1) and soaked in 30 mL of infection medium (INM) (OD600 2.0) for 15 min. The explants were then placed on filter paper to remove excess moisture, transferred to a new culture dish with co-cultivation medium (HRCM), and cultured at 22 °C for 48 h.

After cultivation, the explants were rinsed three times with aseptic water containing 400 mg L*¹ Timentin. After removing excess moisture on aseptic filter paper, the explants were transferred to callus and shoot induction medium (CSIM, Table S1). After 6 d of pre-induction, the explants were transferred into selective pressure medium (SPM, Table S1). After 15-20 d, the regenerated shoots were transferred to shoot elongation medium (SEM, Table S1), and after approximately two weeks, shoots that had grown to 2-3 cm in length were cut and transferred to rooting medium (ROM, Table S1). Root development initiated after one week and continued over the next few weeks. When the roots were strong enough, the plants were maintained at room temperature for 1-2 d before being transplanted in soil.

3. Results

3.1. Development of a hairy root induction system mediated by agr. Rhizogenes strain K599 for gene function analysis

Establishing a reliable induction system for transgenic hairy roots is very useful in screening for efficient gene editing vectors. In this study, we utilized Agr. rhizogenes K599 to evaluate the efficiency of hairy root induction across different jute explant and assessed the expression of pCMV:DsRed2 and pUbi:GFP in the roots (Fig. 1A). Specifically, three types of jute explants (cotyledon node, shoot tip, and hypocotyl) were inoculated with bacterial suspensions at various concentrations (OD600 = 0.5-1.5) for 10-20 min, followed by a 2 d of cultivation in MS solution containing 200 µmol L*¹. The explants were then transferred to rooting medium containing 0.3 mg L*¹ IAA. After 20-30 d, the protocol was optimized based on the number and length of hairy roots, and transgenic roots were confirmed by fluorescence of the reporter genes DsRed2 or GFP (Fig. 1B). Shoot tips, inoculated for 15 min with a bacterial suspension (OD600 = 0.8), exhibited the highest level of hairy root induction after 20 d in rooting medium, with 56% of roots showing positive transgene traits (Figs. S1A, B, S2). Bright red and GFP fluorescence were observed in these roots (Fig. 53). The CcC4H1 gene encodes cinnamate 4-hydroxylase, required for plant cell wall development and lignification [3], and CcWRKY70 is a transcription factor associated with fiber development [13]. Subsequently, bright nuclear and membrane-localized fluorescence, consistent with their subcellular localization in tobacco, was observed in positive transgenic roots of jute overexpressing CCC4H1-GFP and CcWRKY70-GFP (Figs. 1C, S4). Moreover, CcC4H1 overexpression significantly increased root length and altered both cellulose and lignin contents compared to the control (Fig. 1D). Bimolecular fluorescent complementation (BiFC) was used to validate in vivo interactions of multiple genes (Fig. 55). These results proved the development of a hairy root induction system mediated by Agr. rhizogenes K599 in jute, facilitating studies of subcellular localization, protein interaction, and molecular regulation.

3.2. Efficient editing of endogenous genes in jute using a CRISPR/Cas9 system with gRNA driven by the GhU6.7 promoter

U6 promoters from the target species or closely related species are typically used to test gRNA targeting efficiency in using the CRISPR/Cas9 system. Here, we selected two U6 promoters from cotton (Gossypium hirsutum L.) to construct gene editing vectors and evaluate genome mutagenesis efficiency in jute. Two editing vectors targeting exons of CCCLA1 were constructed with two gRNAs driven by GhU6.7 (GhU6.7-gRNA1: 5'-CGTGGAATGAT CAGTGGTTC-3', GhU6.7-gRNA2: 5'-GTTGTCACTGAGAAAGGTCG-3') and GhU6.9 (GhU6.9-gRNA1: 5'-ACTGCCACACTAGATGGACC-3', GhU6.9-gRNA2: 5'-GTTGTCACTGAGAAAGGTCG-3') promoters. These vectors, including a Cas9 gene driven by cotton ubiquitin promoter Ubi and plant-selectable marker for the NeoR/Kank gene driven by the CaMV35S promoter, were constructed as shown in Fig. 1A. The vectors pGhU6.7:gRNA-Ubi:Cas9 and pGhU6.9:gRNA-Ubi:Cas9 were transfected into jute hairy roots by infection with Agr. rhizogenes strain K599. Sixty seven plants with hairy roots were generated in two transformation experiments using pGhU6.7:gRNA-Ubi:Cas9. The specific primers for Cas9 gene detected 30 positive transgenic events (Fig. 1E). Hi-Tom sequencing analysis revealed that 15 samples (including 5 from Sanger sequencing) had base insertion or deletion mutations at target site 2 (gRNA2) (Fig. 1F; Table 1), with a targeted mutagenesis efficiency was 50.0% (15/30). In contrast, the pGhU6.9:gRNA-Ubi:Cas9 vector had a mutagenesis efficiency of 38.5% (5/13) (Table 1). No mutations were found at target site 1 (gRNA1). Thus, the GhU6.7 promoter achieved efficient gene editing.

3.3. Generation of stable transformation for editing CcCLA1 by Agrobacterium-mediated CRISPR/Cas9 to produce stably transformed albinic seedlings

To introduce pGhU6.7:gRNA(CcCLA1)-Ubi:Cas9 plasmid into jute using an Agrobacterium-mediated stable transformation method, several essential factors were optimized based on previous research in our laboratory [12]. These factors included using Agr. tumefaciens strain GV3101 and adjusting hormone ratios in the callus and shoot induction medium (CSIM), shoot elongation medium (SEM), and rooting medium (ROM) (Table S1). In this study, the explants were infected for 15 min, cultivated for 48 h, and then subjected to callus and shoot induction (15–20 d), shoot elongation (10–15 d), and root induction (10–15 d). Regenerated seedlings were obtained after one day at room temperature (Fig. 1G). Approximately 830 seedlings with bud clumps were produced for stable transformation. Two albinic seedlings (#1# and #2#, Fig. 1H) were confirmed to contain the Cas9 gene using specific primers P-Cas9-F/R (Fig. S6; Table S3). Screening under selective pressure medium (SPM, Table S1) increased the transgenic efficiency of the Cas9 gene in regenerated seedlings to 90.5% (19/21) (Fig. S6; Table S2).

PCR using specific primer pair P-CLA1-F/R (Table S3) amplified a 721 bp fragment encompassing two target sites in the CcCAL1 gene in plants #1# and #2#. Sanger sequencing revealed a single base deletion at the gRNA2 target site in each plant (Fig. 1I), with no mutation observed at either target site in green plants. Overall, the gene editing efficiency of the Agrobacterium-mediated stable genetic transformation system for jute was about 5.3% (Table S2). These results demonstrated that the CRISPR/Cas9 system can induce targeted knockout of the CcCLA1 gene and generate stable phenotypic changes in jute.

4. Discussion

Transgenic hairy roots are known for their stable genetic traits and rapid proliferation, making them widely used in gene function analysis and production of plant secondary metabolites [14–16]. Although Agr. rhizogenes strain LBA1334 was known to induce transgenic hairy roots in jute, induction efficiency was low and induction time was prolonged [17]. In this study, Agr. rhizogenes strain K599 proved more suitable for inducing hairy root formation in jute, producing abundant numbers of transgenic plants with hairy roots in approximately 20 d. This system showed potential for functional studies of genes such as CcC4H1 and CcWRKY70, facilitating analysis of gene interaction and regulation, subcellular localization, protein-nucleic acid interactions, and gene editing.

Developing a stable and efficient genetic transformation system for jute has been a priority for a considerable time. Researchers attempted to regenerate shoots from various explants [18,19], with significant advancements particularly in shoot regeneration from shoot tips and cotyledonary nodes [10,11,20]. Saha et al. [21,22] were the first to report stable transformation events using shoot tips, subsequently optimizing the system over time, and other studies followed [11]. In this study, we further optimized infection and germination efficiency using cotyledonary node explants.

CRISPR/Cas9 genome editing technology has become widely used in functional genomics and precise molecular breeding [23]. However, there are few studies on genome editing of jute. Efficient genome editing relies on gRNA driven by a suitable U6 promoter [24]. Here, we identified cotton promoter GhU6.7 to be effective for genome editing in jute using the transgenic hairy root system, and achieved an editing efficiency of 50.0%. Following extensive screening of stable transformants, we obtained gene-edited plants by targeting the CcCLA1 gene. Although the mutagenesis efficiency of the GhU6.7-guided gRNA was higher than that of GhU6.9, the efficiency was not optimal, especially at target site 1 (gRNA 1) of the CcCLA1 gene, where no reliable editing events was detected. Nevertheless, the transgenic hairy root induction system developed in the study provided a tool for testing gRNA targeted efficiency and produced genome-edited plants. This gene editing system producing stable transformation in jute offers a reliable means for future gene function research and will allow manipulation of target genes to improve important agronomic traits [5]. Multi-gene editing will enable the aggregation of multiple superior alleles in non-transgenic plants leading to new germplasm and varieties of jute.

CRediT authorship contribution statement

Shaolian Jiang: Data curation, Formal analysis, Investigation, Methodology. Qin Li: Investigation, Visualization, Writing – original draft, Writing – review & editing. Xiangxue Meng: Investigation, Methodology. Mengxin Huang: Investigation, Methodology. Jiayu Yao: Investigation, Methodology. Chuanyu Wang: Investigation. Pingping Fang: Resources. Aifen Tao: Resources. Jiantang Xu: Resources. Jianmin Qi: Resources. Shuangxia Jin: Methodology, Resources. Liwu Zhang: Funding acquisition, Methodology, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31771369), the Natural Science Foundation of Fujian, China (2023J01443), and the China Agriculture Research System of the Ministry of Agriculture and MARA (CARS-16). We thank Tao Lan for providing vectors of OsSPL10 and OsWOX3B, and Muhammad Zohaib Afzal for English editing of the manuscript.

Appendix A. Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2024.06.002.

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ARTICLE INFO

Article history:

Received 8 December 2023

Revised 11 June 2024

Accepted 12 June 2024

Available online 10 July 2024

Footnote

* Corresponding authors.

E-mail addresses: [email protected] (S. Jin), [email protected] (L. Zhang).

1 The authors contributed equally to this work.

References

References

[1] A. Kundu, N. Topdar, D. Sarkar, M.K. Sinha, A. Ghosh, S. Banerjee, M. Das, H.S. Balyan, B.S. Mahapatra, P.K. Gupta, Origins of white (Corchorus capsularis L.) and dark (C. olitorius L.) jute: a reevaluation based on nuclear and chloroplast microsatellites, J. Plant Biochem. Biotechnol. 22 (2013) 372–381.

[2] A. Chakraborty, D. Sarkar, P. Satya, P.G. Karmakar, N.K. Singh, Pathways associated with lignin biosynthesis in lignomaniac jute fibres, Mol. Genet. Genomics 290 (2015) 1523–1542.

[3] L.L. Zhang, X.K. Ma, X.T. Zhang, Y. Xu, A.K. Ibrahim, J.Y. Yao, H.X. Huang, S. Chen, Z.Y. Liao, Q. Zhang, S. Niyitanga, J.X. Yu, Y. Liu, X.M. Xu, J.J. Wang, A.F. Tao, J.T. Xu, S.Y. Chen, X. Yang, Q.Y. He, L.H. Lin, P.P. Fang, L.M. Zhang, R. Ming, J.M. Qi, L. W. Zhang, Reference genomes of the two cultivated jute species, Plant Biotechnol. J. 19 (2021) 2235–2248.

[4] Y. Huang, H.R. Dong, M.Q. Shang, K.J. Wang, CRISPR/Cas systems: The link between functional genes and genetic improvement, Crop J. 9 (2021) 678–687.

[5] J. Luo, S. Li, J. Xu, L. Yan, Y. Ma, L. Xia, Pyramiding favorable alleles in an elite wheat variety in one generation by CRISPR-Cas9-mediated multiplex gene editing, Mol. Plant 14 (2021) 847–850.

[6] A.H. Khan, Y.Z. Ma, Y.L. Wu, A. Akbar, M. Shaban, A. Ullah, J.W. Deng, A.S. Khan, H.B. Chi, L.F. Zhu, X.L. Zhang, L. Min, High-temperature stress suppresses allene oxide cyclase 2 and causes male sterility in cotton by disrupting jasmonic acid signaling, Crop J. 11 (2023) 33–45.

[7] Y.Z. Chen, M.C. Fu, H. Li, L.G. Wang, R.Z. Liu, Z.J. Liu, X.L. Zhang, S.X. Jin, Higholeic acid content, nontransgenic allotetraploid cotton (Gossypium hirsutum L.) generated by knockout of GhFAD2 genes with CRISPR/Cas9 system, Plant Biotechnol. J. 19 (2021) 424–426.

[8] P.C. Wang, J. Zhang, L. Sun, Y.Z. Ma, J. Xu, S.J. Liang, J.W. Deng, J.F. Tan, Q.H. Zhang, L.L. Tu, H. Daniell, S.X. Jin, X.L. Zhang, High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system, Plant Biotechnol. J. 16 (2018) 137–150.

[9] Q. Liao, X. Cheng, T. Lan, X. Guo, Z. Su, X. An, Y. Zheng, H. Cui, W. Wu, T.J.T.C.J. Lan, OsSPL10 controls trichome development by interacting with OsWOX3B at both transcription and protein levels in rice (Oryza sativa L.), Crop J. 11 (2023) 1711–1718.

[10] T. Saha, M. Ghosh, S.K. Sen, Plant regeneration from cotyledonary explants of jute, Corchorus capsularis L., Plant Cell Rep. 18 (1999) 544–548.

[11] B. Pushyami, M.R. Beena, M.K. Sinha, P.B. Kirti, In vitro regeneration and optimization of conditions for Agrobacterium mediated transformation in jute, Corchorus capsularis, J. Plant Biochem. Biotechnol. 20 (2011) 39–46.

[12] G. Zhang, Y. Zhang, J. Xu, F. Li, A. Tao, L. Zhang, P. Fang, L. Lin, J. Qi, An efficient regeneration system and optimization of the transformation from the cotyledonary node of jute (Corchorus capsularis L.), J. Nat. Fibers 12 (2015) 303–310.

[13] L. Zhang, X. Wan, Y. Xu, S. Niyitanga, J. Qi, L. Zhang, De novo assembly of transcriptome and genome-wide identification reveal GA3 stress-responsive WRKY transcription factors involved in fiber formation in jute (Corchorus capsularis), BMC Plant Biol. 20 (2020) 403.

[14] M. Li, Z. Hu, Q.Y. Jiang, X.J. Sun, Y. Guo, J.C. Qi, H. Zhang, GmNAC15 overexpression in hairy roots enhances salt tolerance in soybean, J. Integr. Agric. 17 (2018) 530–538.

[15] M.C. Vaccaro, M. Alfieri, N. De Tommasi, T. Moses, A. Goossens, A. Leone, Boosting the synthesis of pharmaceutically active abietane diterpenes in S. sclarea hairy roots by engineering the GGPPS and CPPS Genes, Front. Plant Sci. 11 (2020) 924.

[16] S. Singh, P. Pandey, M.Q. Akhtar, A.S. Negi, S. Banerjee, A new synthetic biology approach for the production of curcumin and its glucoside in Atropa belladonna hairy roots, J. Biotechnol. 328 (2021) 23–33.

[17] T. Chattopadhyay, S. Roy, A. Mitra, M.K. Maiti, Development of a transgenic hairy root system in jute (Corchorus capsularis L.) with gusA reporter gene through Agrobacterium rhizogenes mediated co-transformation, Plant Cell Rep. 30 (2011) 485–493.

[18] A.M. Abbas, J.K. Jones, P.D.S. Caligari, Clonal propagation by in vitro culture of Corchorus (jute), Plant Cell Tiss. Organ Cult. 47 (1997) 231–238.

[19] P. Bharadwaj, M.R. Beena, M.K. Sinha, P.B. Kirti, Studies on explant regeneration and protoplast culture from hypocotyl segments of jute, Corchorus capsularis L., J. Plant Stud. 2 (2012) 27.

[20] R.H. Sarker, G.M. Al-Amin, M.I. Hoque, In vitro regeneration in three varieties of white jute (Corchorus capsularis L.), Plant Tissue Cult. Biotech. 17 (2007) 1118.

[21] P. Saha, K. Datta, S. Majumder, C. Sarkar, S.P. China, S.N. Sarkar, D. Sarkar, S.K. Datta, Agrobacterium mediated genetic transformation of commercial jute cultivar Corchorus capsularis cv. JRC 321 using shoot tip explants, Plant Cell Tiss. Organ Cult. 118 (2014) 313–326.

[22] S. Majumder, C. Sarkar, P. Saha, B.S. Gotyal, S. Satpathy, K. Datta, S.K. Datta, Bt jute expressing fused d-endotoxin Cry1Ab/Ac for resistance to Lepidopteran pests, Front. Plant Sci. 8 (2017) 2188.

[23] C. Gao, Genome engineering for crop improvement and future agriculture, Cell 184 (2021) 1621–1635.

[24] C. Ren, Y. Liu, Y. Guo, W. Duan, P. Fan, S. Li, Z. Liang, Optimizing the CRISPR/ Cas9 system for genome editing in grape by using grape promoters, Hortic. Res. 8 (2021) 52.